Characterization of RNAs captured through a new engineered protein-mediated affinity purification

We generated a new recombinant protein HARD (High-affinity RNA-binding Domain) with non-sequence-specific high-affinity on RNAs and developed the immobilized-HARD protein-mediated RNA affinity purification approach (HARD-AP).We showed that HARD-AP successfully captured all RNA biotypes. Overall design: We incubated the HARD beads (chemically conjugate the HARD protein onto agarose resin) with the total RNA of HEK293 cells and then purified RNAs left on beads after stringently washing.We performed Ribo-Zero RNA sequencing (RNA-seq) of the HARD beads-bound RNAs and input RNAs to test the nature of the captured RNAs.